Approximately one-third of all newly synthesized proteins in eukaryotes enter the endoplasmic reticulum (ER), a compartment in which specialized machinery exists to support the post-translation modification of polypeptides and to facilitate protein folding. Nevertheless, a significant proportion of many secreted proteins fold inefficiently. This problem is particularly evident for integral membrane proteins, given that the native conformations of these topologically complex species must be achieved in the ER lumen, within the ER membrane, and in the cytoplasm. In the event that folding is delayed or aborted, the resulting polypeptide may be selected and then targeted for degradation by the cytoplasmic proteasome. This process has been termed ER associated degradation (ERAD) and can be sub-divided into the following steps: substrate recognition, retro-translocation or dislocation (delivery to the cytoplasm), ubiquitin conjugation, and degradation. Because many membrane proteins are essential for cellular and organismal homeostasis, it is not surprising that a growing number of ERAD substrates have been linked to human disease. In order to define the pathway by which ERAD substrates are ultimately destroyed, model substrates were designed to test specific hypotheses and novel in vitro assays were developed in which each step during the degradation pathway can be examined. These approaches have been empowered by the use of reagents prepared from the yeast S. cerevisiae, which permits the use of components isolated from wild type or mutant strains. Therefore, factors that catalyze each step during ERAD can be isolated, characterized, and tested in complementary in vitro and in vivo systems. The questions asked in this application include: How is an ATP-requiring engine, which helps extract ERAD substrates from the membrane regulated by associated factors? How is a cytoplasmic, misfolded domain recognized, retro-translocated, and destroyed when the hydrophobicity of the membrane anchor is altered, or when the domain is linked to the membrane by a lipid? Do different factors act on an ERAD substrate when the domain is positioned in the ER lumen versus cytoplasm? And, how are transmembrane domains retained in solution after substrate retro-translocation? Answers to these questions will further the applicant's long-term goal to modulate the ERAD pathway in order to off-set the catastrophic consequences of ERAD-associated diseases. PUBLIC HEALTH RELEVANCE: The misfolding and degradation of integral membrane proteins can cause specific diseases, including cystic fibrosis, hyper- and hypo-tension, and diabetes. The experiments proposed in this application seek to define how recently identified factors impact the degradation of membrane proteins. Experiments will be performed in a model organism and in complementary biochemical systems. The knowledge gained from these studies may lead to the development of novel therapies and provide methods to define the pathway of disease onset.